Abstract 2,5- Diketo- d-threo- hexonic acid (2,5-diketo- d-gluconic acid, 1), a crucial intermediate in the microbial production of l-threo- hex-2-enono-1,4-lactone ( l-ascorbic acid, vitamin C), was isolated from the fermentation broth of bacterium Erwinia citreus ATCC 31623, and its regio- and stereo-selective hydrogenation, catalyzed by the water-soluble Ru(II) complex of tris( m-sulfophenyl)phosphine (TPPTS), was performed. The effect of hydrogen pressure, temperature, pH, and catalyst-to-substrate ratio on regio- and stereo-selectivity of the process was studied. At low pH, over 90% regioselectivity in favor of the reduction of the 5-keto group in 1 was achieved, affording l-xylo-2- hexulosonic acid (2-keto- l-gulonic acid, 2) as the main product. Maximal diastereoselectivity, i.e. ratio between 2 and 2-keto- d-gulonic acid (3) expressed as diastereomeric excess (d.e.%), amounted to ca. 50% and was not influenced by any of the above reaction parameters.